Method Article

Light-Controlled Fermentations for Microbial Chemical and Protein Production

DOI:

10.3791/63269

⸱

March 22nd, 2022

 ,  , 
1Department of Chemical and Biological Engineering, Princeton University, 2The Andlinger Center for Energy and the Environment, Princeton University, 3Department of Molecular Biology, Princeton University, 4High Meadows Environmental Institute, Princeton University

* These authors contributed equally

In This Article

Summary

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Optogenetic control of microbial metabolism offers flexible dynamic control over fermentation processes. The protocol here shows how to set up blue light-regulated fermentations for chemical and protein production at different volumetric scales.

Abstract

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Microbial cell factories offer a sustainable alternative for producing chemicals and recombinant proteins from renewable feedstocks. However, overburdening a microorganism with genetic modifications can reduce host fitness and productivity. This problem can be overcome by using dynamic control: inducible expression of enzymes and pathways, typically using chemical- or nutrient-based additives, to balance cellular growth and production. Optogenetics offers a non-invasive, highly tunable, and reversible method of dynamically regulating gene expression. Here, we describe how to set up light-controlled fermentations of engineered Escherichia coli and Saccharomyces cerevisiae for the production of chemicals or recombinant proteins. We discuss how to apply light at selected times and dosages to decouple microbial growth and production for improved fermentation control and productivity, as well as the key optimization considerations for best results. Additionally, we describe how to implement light controls for lab-scale bioreactor experiments. These protocols facilitate the adoption of optogenetic controls in engineered microorganisms for improved fermentation performance.

Introduction

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Optogenetics, the control of biological processes with light-responsive proteins, offers a new strategy to dynamically control microbial fermentations for chemical and protein production1,2. The burden of engineered metabolic pathways and the toxicity of some intermediates and products often impairs cell growth3. Such stresses can lead to poor biomass accumulation and reduced productivity3. This challenge can be addressed by temporally dividing fermentations into a growth and production phase, which devote metabolic resources to biomass accumulation or product sy....

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Protocol

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1. Light-controlled chemical production using the S. cerevisiae OptoINVRT7 circuit

  1. Strain construction
    1. Obtain a strain with his3 auxotrophy, as this marker is necessary for most existing OptoINVRT plasmids5. If seeking optogenetic regulation of a gene that is native to S. cerevisiae, construct a strain in which any endogenous copy of the gene is deleted.
    2. Linearize the plasmid containing the OptoINVRT7 circuit, such as EZ-L4395, and integrate it into the his3-locus of the auxotrophic strain using standard lithium-acetate transfor....

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Results

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Optogenetic regulation of microbial metabolism has been successfully implemented to produce a variety of products, including biofuels, bulk chemicals, proteins, and natural products5,6,7,12,13. Most of these processes are designed for cell growth to occur in the light (when low cell density poses minimal challenges with light penetration), and for production t.......

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Discussion

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Dynamic control has long been applied to improve yields for metabolic engineering and recombinant protein production4. Shifts in enzymatic expression are most typically implemented using chemical inducers such as IPTG21, galactose22, and tetracycline23, but have also been mediated using process conditions such as temperature and pH. Optogenetic control of gene expression eliminates the need for changes to fermentation paramete.......

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Disclosures

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The authors have applied for several patents for the optogenetic circuits and methods described in this article.

Acknowledgements

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This research was supported by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research Award Number DE-SC0019363, the NSF CAREER Award CBET-1751840, The Pew Charitable Trusts, and the Camille Dreyfus Teacher-Scholar Award.

....

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Light-controlled chemical production using S. cerevisiae
24-well culture plateUSA ScientificCC7672-7524
Agar powderThermo Fisher Scientific303991049
Aluminum foilReynoldsB004NG90YO
BioSpectrometer with μcuvetteEppendorf6135000923
Blue LED panelHQRP884667106091218
EZ-L439 OptoINVRT7 PlasmidN/AN/ASee Reference 1
GlucoseThermo Fisher Scientific501879892 (G8270-5KG)
MicrocentrifugeThermo Fisher Scientific75002403
Microcentrifuge tubesUSA Scientific1615-5510
Orbital ShakerYamato Scientific AmericaSOU-300
Petri dishCelltreat229656
PmeINew England BiolabsR0560L
Quantum meterApogee InstrumentsMQ-510
Replica-plating deviceThomas ScientificF37848-0000
Replica-plating padsSunrise Science Products3005-012
SC-His powderSunrise Science Products1303-030
SC Complete powderSunrise Science Products1459-100
Sterile sealing filmExcel ScientificSTR-SEAL-PLT
YPD agar platesVWR100217-054
ZeocinThermo Fisher ScientificR25005
Light-controlled protein production using E. coli
6X SDS Sample BufferCepham Life Sciences10502
12% Acrylamide protein gelsThermo Fisher ScientificNP0341BOX
24-well culture plateUSA ScientificCC7672-7524
Aluminum foilReynoldsB004NG90YO
BioSpectrometer with μcuvetteEppendorf6135000923
Blue LED panelHQRP884667106091218
Coomassie Brilliant Blue G-250Thermo Fisher Scientific20279
Electrophoresis cellBio-Rad1658004
Electrophoresis power supplyBio-Rad1645050
LB broth (Miller)Fisher ScientificBP97235
MicrocentrifugeThermo Fisher Scientific75002403
Microcentrifuge tubesUSA Scientific1615-5510
NaClThomas ScientificSX0425-1
OptoLAC plasmidsN/AN/ASee Reference 2
Orbital ShakerYamato Scientific AmericaSOU-300
Petri dishCelltreat229656
Quantum meterApogee InstrumentsMQ-510
SOC mediumThermo Fisher Scientific15544034
ThermomixerEppendorf5382000015
Tris baseFisher ScientificBP1521
Three-phase fermentation using S. cerevisiae
Same materials as "Light-controlled chemical production using S. cerevisiae" protocol plus the following:
EZ-L580 OptoAMP4 PlasmidN/AN/ASee Reference 10
Chemical production in a light-controlled bioreactor
Aluminum foilReynoldsB004NG90YO
AntifoamSigma-AldrichA8311
Bioreactor with control stationEppendorfB120110001
BioSpectrometer with μcuvetteEppendorf6135000923
BleachVWR Scientific89501-620 (CS)
Blue LED panelHQRP884667106091218
BPT tubingFisher Scientific14-170-15
GlucoseThermo Fisher Scientific501879892 (G8270-5KG)
Hydrochloric acid (HCl)Fisher Scientific7647-01-0
M9 Minimal SaltsThermo Fisher ScientificA1374401
MicrocentrifugeThermo Fisher Scientific75002403
Microcentrifuge tubesUSA Scientific1615-5510
NH4OH SolutionSigma-AldrichI0503-1VL
Orbital ShakerYamato Scientific AmericaSOU-300
Quantum meterApogee InstrumentsMQ-510
SC Complete powderSunrise Science Products1459-100

References

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  1. Figueroa, D., Rojas, V., Romero, A., Larrondo, L. F., Salinas, F. The rise and shine of yeast optogenetics. Yeast. 38 (2), 131-146 (2021).
  2. Pouzet, S., et al. The promise of optogenetics for bioproduction: Dynamic control strategies and scale-up instruments. Bioengi....

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Tags

Light Controlled FermentationOptogenetic RegulationMicrobial Cell FactoriesRecombinant Protein ProductionEscherichia ColiSaccharomyces CerevisiaeDynamic Gene ExpressionBioreactor ExperimentsPulsed Light InductionChemical Production

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